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Koch2010
1. Int. J. Mol. Sci. 2011, 12, 39-45; doi:10.3390/ijms12010039
OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article
Erwinia mallotivora sp., a New Pathogen of Papaya (Carica
papaya) in Peninsular Malaysia
Noriha Mat Amin *, Hamidun Bunawan, Rohaiza Ahmad Redzuan and Indu Bala S. Jaganath
Biotechnology Research Centre, Malaysian Agricultural Research and Development Institute,
P.O Box 12301, General Post Office, 50774 Kuala Lumpur, Malaysia;
E-Mails: hamidunb@yahoo.com (H.B.); rohaiza@mardi.gov.my (R.A.R.);
indu@mardi.gov.my (I.B.S.J.)
* Author to whom correspondence should be addressed; E-Mail: noriha@mardi.gov.my;
Tel.: +603-8943-7563; Fax: +603-8945-7630.
Received: 3 November 2010; in revised form: 10 December 2010 / Accepted: 20 December 2010 /
Published: 24 December 2010
Abstract: Erwinia mallotivora was isolated from papaya infected with dieback disease
showing the typical symptoms of greasy, water-soaked lesions and spots on leaves.
Phylogenetic analysis of 16S rRNA gene sequences showed that the strain belonged to the
genus Erwinia and was united in a monophyletic group with E. mallotivora DSM 4565
(AJ233414). Earlier studies had indicated that the causal agent for this disease was
E. papayae. However, our current studies, through Koch’s postulate, have confirmed that
papaya dieback disease is caused by E. mallotivora. To our knowledge, this is the first new
discovery of E. mallotivora as a causal agent of papaya dieback disease in Peninsular
Malaysia. Previous reports have suggested that E. mallotivora causes leaf spot in Mallotus
japonicus. However, this research confirms it also to be pathogenic to Carica papaya.
Keywords: Carica papaya; Erwinia mallotivora; papaya dieback
1. Introduction
The genus Erwinia is classified in the family of Enterobactericeae, typically facultative anaerobic,
gram negative rods possessing peritrichious flagella. Most members of this genus are naturally
plant-pathogenic and plant-associated bacteria. Erwinia sp. are responsible for such basic types of plant
2. Int. J. Mol. Sci. 2011, 1 40
disease as necrosis, blight, soft rot and wilt in a number of crop plants. For example, fire blight caused
by E. amylovora is the most destructive disease in pear and apple orchards in many parts of world [1].
E. chrysanthemi pv. zeae and E. corotovora subsp. carotovora cause a highly destructive disease in
maize and soft rot of potatoes as well as a wide range of fruits and vegetables [2].
Papaya (Carica papaya) is an economically important fruit crop grown in Malaysia with an export
value of about RM100–120 million per year [3]. Currently, one of the major threats of this industry in
Malaysia is papaya dieback disease. The common symptoms observed include greasy, water soaked
lesions and spots on leaves, as well as foliar and angular lesions. These lesions can lead to secondary
infection, which can eventually cause the death of the papaya plant. This disease has been a problem to
papaya growers for almost a decade, destroying more than one million plants.
The disease was first identified in Malaysia near Batu Pahat, Johor in late 2003. Another incidence
was later reported in Bidor, Perak, in October 2004. More recently, Maktar et al. [4] reported
E. papayae as causing papaya dieback in Malaysia. However, they performed no significant
biochemical tests to distinguish E. papayae and E. mallotivora, which are closely related species. They
relied on two basic biochemical tests (oxidase and catalase) and the sequence similarity of the 16S
rRNA gene to confirm E. papayae as a causal agent of papaya dieback in Malaysia. E. papayae was
first reported by Gardan et al. [5] as the causal organism of papaya bacterial canker in the Caribbean
region. However, the advanced stage of papaya dieback reported by Maktar et al. [4] had no canker
symptom. Thereafter, no additional information on further occurrence of the disease has been reported
until our finding, reported here, of E. mallotivora as a causal organism for this disease.
The aim of this study was, firstly, to isolate and identify the papaya dieback pathogen, and
subsequently, to confirm its pathogenicity through Koch’s postulate. By combining phenotypic and
genotypic information, we confirmed E. mallotivora as the causal agent of papaya dieback disease in
Peninsular Malaysia.
2. Results and Discussion
Gram-negative and rod-shaped bacteria with a peritrichous flagella arrangement were consistently
isolated from water-soaked lesions (Figure 1). The isolate from infected tissues produced hyaline
colonies on Luria Bertani (LB) agar, typically with slow growth (2–3 days incubation). Colonies were
creamy to white in color on King’s B [6] agar after 2–3 days incubation and on YBGA (0.7% yeast
extract, 0.7% bactopeptone, 0.7% glucose and 1.5% agar, pH 7.2) medium after 3–4 days incubation at
25 ° No water-soluble or non-water-soluble pigments were produced on King’s B medium.
C.
Biochemical analysis revealed that the strains are catalase positive and oxidase negative with the ability
to form reducing compounds from sucrose. In addition, the strains produced acid from D-mannitol and
utilized both lactate isomers, D- and L-lactate, which is consistent with the results reported by Goto [7]
and Gardan et al. [5] for E. mallotivora. In contrast, E. papayae was unable to produce acid from
mannitol, DL-lactate and sucrose-reducing compounds [5]. The apparent phenotypic characteristics of
E. papayae and E. mallotivora are summarized in Table 1.
3. Int. J. Mol. Sci. 2011, 1 41
Figure 1. Transmission electron microscope image of the strain of E. mallotivora isolated
from dieback-infected papaya tree.
1μm
Table 1. Distinguishable characteristics of E. mallotivora and E. papaya.
Characteristic E. mallotivora (BT-Mardi) E. mallotivora [7] E. papaya [5]
Blue pigment on King’s B agar − − +
Citrate utilization + + −a
Reducing substances from sucrose + + −
D-Mannitol + + −
L-Arabinose − − +
a
More than 70% of the strains negative.
Pathogenicity testing showed that the injected isolates formed brown spots on the leaves, water
soaked lesions and greasy spots on the stem after 4–5 days of inoculation around the sites of
inoculation and death of the plant occurred about 15 days after inoculation (Figure 2A, 2B). These
symptoms observed are typical of those found in natural papaya dieback infection. Control plants
showed no symptoms of the disease. E. mallotivora was recovered from the lesions as pure cultures,
supporting our preliminary results that suggest that this bacterium is responsible for papaya dieback
disease; the same pathogen has been reported to infect M. japonicus with only mild symptoms
exhibiting as small lesions on the stem but not causing dieback of the infected shoot [7]. However,
with papaya, E. mallotivora showed severe symptoms and caused dieback of the infected shoot,
leading to the destruction of the plants without evidence of canker symptom.
Erwinia sp. have been long known as plant pathogens that produce a wide range of enzymes able to
degrade plant cell wall components [8,9]. Leu et al. [10] reported that Erwinia cypripedii caused black
rot on seedlings, trees and fruits of papaya in Taiwan. In 1982, Trujillo and Schroth reported two
diseases. Erwinia decline of papaya (D strains) and the Erwinia mushy canker disease of papaya (MC
strains), in Hawaii [11]. Webb [12] reported a species of Erwinia that caused angular water-soaked
lesions on leaves and firm water-soaked cankers on the stems of papaya. Recently, the causal agent of
bacterial canker in papaya was identified as a new bacterium species named E. papayae [5]. In this
4. Int. J. Mol. Sci. 2011, 1 42
study, we report for the first time E. mallotivora as a new pathogen of papaya. The disease is
transmitted from plant to plant or to other parts of the same plants, primarily by insects, birds, humans
and rain splash [4,13]. Bacteria may enter through stomata, hydathodes, and lenticals and through
wounds made by, for example, insects and hailstorms. From the leaf, the bacteria pass into the petiole
and the stem, first colonizing and then moving through vessels of the plant.
Figure 2. Papaya dieback symptoms caused by E. mallotivora (A) Leaf spots formed along
the main vein of infected leaf (left) compared to a healthy leaf (negative control: right);
(B) Greasy and water-soaked lesions leading to the destruction of papaya tree (Arrow).
A BLAST analysis of the 1.5-kb 16S rRNA gene sequence showed high nucleotide identity (99%)
to the 16S rRNA gene of E. mallotivora DSM 4565 (accession number AJ233414) and E. papayae
(accession number AY131237). Phylogenetic analysis revealed that the E. mallotivora (BT-MARDI)
from Malaysia (accession number HQ456230) belongs to the genus Erwinia and is united in a
monophyletic group with E. mallotivora DSM 4565 and E. papayae. It showed that E. mallotivora and
E. papayae are closely related species [5]. The group was supported with a high number of bootstraps
in a neighbor-joining (NJ) tree (Figure 3). E. mallotivora (BT-MARDI) formed a robust cluster with
the strain E. mallotivora (AJ233414).
5. Int. J. Mol. Sci. 2011, 1 43
Figure 3. Phylogenetic tree based on neighbor-joining phylogram.
E.mallotivora (BT-MARDI)
E.mallotivora (AJ233414)
E.papayae (AY131237)
E.psidii (Z96085)
E.persicinus (U80205)
E.pyrifoliae (AJ009930)
E.amylovora (U80195)
E.cypripedii (U80201)
E.tracheiphila (Y13250)
3. Experimental Section
3.1. Bacterial Isolation
The strain was isolated from water-soaked lesions formed on the leaves, stems and fruits of
naturally infected papaya plants at MARDI Research Station Serdang, Selangor, in early August 2010.
Isolation procedures followed those of Agrios [14].
3.2. Koch’s Postulate
For pathogenicity testing, three month-old seedlings of C. papaya (cv. Sekaki) were infected with
the bacterial suspension to determine the causative agent. Leaves of experimental plants were
inoculated by pricking their abaxial surface with a sterile needle and injection with 50 μL of the
inoculate suspension at a concentration of 1 × 108 CFU per mL into the apex of the seedlings,
according to standard techniques described by [15]. Control plants were similarly inoculated with
sterile water. Plants were incubated under greenhouse conditions at temperatures between 28 ° and
C
32 ° in the day and 25 ° and 28 ° at night. Using the same technique, bacteria were reisolated once
C C C
symptoms appeared. Selected biochemical study of this bacteria was performed according to Gardan
et al. [5,16] and the bacterial strain was identified using the API 20E system (BioMé rieux, USA).
Negative staining protocols for transmission electron microscopy were carried out as described by Lee
and Taylor [17].
3.3. PCR Amplification and Cloning of 16S rRNA Gene
High quality DNA was isolated using a GenEluteTM Bacterial Genomic DNA Extraction Kit
according to the manual procedures (Sigma-Aldrich, USA). PCR amplification was performed in a
25 L reaction using thermostable DyNAzymeTM EXT DNA polymerase (Finnizymes, Finland) in a
PTC-200 thermal cycler (MJ Research, USA). The reaction mixture consisted of 1 × PCR buffer;
2.0 mM of MgCl2, 0.2 mM of dNTPs, 2 M of forward and reverse primers, 100 ng of bacterial
genomic DNA as template and 2.5 U of the enzyme mix. The 16S rRNA gene was amplified with the
6. Int. J. Mol. Sci. 2011, 1 44
universal F8 (5'-AGAGTTTGATCMTGGCTC-3') and rP2 (5'-ACGGCTACCTTGTTACGACTT-3')
primer pair [18]. Amplification reaction was carried out with the following cycling conditions: primary
denaturation for 3 min at 95 ° followed by 30 cycles of 30 s at 94 ° 1 min at 55 ° and 2 min of
C, C, C
72 ° and a final extension of 10 min at 72 ° The PCR product was run through a 1% agarose gel
C, C.
and purified using QIA Quick Gel Extraction Kit (Qiagen, Valencia, CA, U.S.). The TOPO TA
Cloning® Kit (Invitrogen, U.S.) was used for cloning of the PCR product following the manufacturer’s
protocol. Minipreparation of plasmid DNA was carried out using the QIAprep Spin miniprep kit
(Qiagen, Valencia, CA, U.S.), followed by restriction endonuclease analysis to identify the clones for
sequencing. DNA sequencing was performed using an ABI Prism Dye Terminator Cycle Sequencing
Ready Reaction kit and ABI PRISM 3100 Genetic Analyzer (Perkin-Elmer, Foster City, CA) following
the manufacturer’s instructions. The 16S rRNA gene sequences were aligned using ClustalW and
analyzed by neighbor-joining (NJ) using the MEGA 4 program [19].
4. Conclusions
Our phenotypic observations, biochemical analysis and genetic studies lead us to conclude that the
isolated strain belonged to the E. mallotivora species and is the main causal agent of papaya dieback
disease in Peninsular Malaysia. This identification is critical not only for further understanding of the
pathogenic mechanism, but concurrently for development of suitable control strategies, including
selection and development of resistant varieties to overcome the disastrous effect of this disease.
Acknowledgements
This research was supported by the Ministry of Agriculture, Malaysia under the research grant
05-03-08-SF1049. We thank Khairun Hisam Nasir for assisting us in the sample collection.
References
1. Gwyne, D.C. Fire blight in perry pears and cider apples in the South West of England. Acta Hortic.
1984, 151, 41–47.
2. Reifschneider, F.J.B.; Lopes, C.A. Bacterial top and stalk rot of maize in Brazil. Plant Dis. 1982,
66, 519–520.
3. Rabu, M.R.; Mat Lin, R. Prospect of papaya in the world market: Malaysia perspective. In
Proceeding of First International Symposium on Papaya, Genting Higlands, Malaysia,
22–24 November 2005.
4. Maktar, N.H.; Kamis, S.; Mohd Yusof, F.Z.; Hussain, N.H. Erwinia papayae causing papaya
dieback in Malaysia. Plant Pathol. 2008, 57, 774.
5. Gardan, L.; Christen, R.; Achouak, W.; Prior, P. Erwinia papayae sp. nov., a pathogen of papaya
(Carica papaya). IJSEM 2004, 54, 107–113.
6. King, E.O.; Ward, M.K.; Raney, D.E. Two simple media for the demonstration of pyocyanin and
fluorescin. J. Lab. Clin. Med. 1954, 44, 301–307.
7. Goto, M. Erwinia mallotivora sp. nov., the causal organism of bacterial leaf spot of Mallotus
japonicus. Int. J. Syst. Bacteriol. 1976, 26, 467–473.
![Int. J. Mol. Sci. 2011, 1 40
disease as necrosis, blight, soft rot and wilt in a number of crop plants. For example, fire blight caused
by E. amylovora is the most destructive disease in pear and apple orchards in many parts of world [1].
E. chrysanthemi pv. zeae and E. corotovora subsp. carotovora cause a highly destructive disease in
maize and soft rot of potatoes as well as a wide range of fruits and vegetables [2].
Papaya (Carica papaya) is an economically important fruit crop grown in Malaysia with an export
value of about RM100–120 million per year [3]. Currently, one of the major threats of this industry in
Malaysia is papaya dieback disease. The common symptoms observed include greasy, water soaked
lesions and spots on leaves, as well as foliar and angular lesions. These lesions can lead to secondary
infection, which can eventually cause the death of the papaya plant. This disease has been a problem to
papaya growers for almost a decade, destroying more than one million plants.
The disease was first identified in Malaysia near Batu Pahat, Johor in late 2003. Another incidence
was later reported in Bidor, Perak, in October 2004. More recently, Maktar et al. [4] reported
E. papayae as causing papaya dieback in Malaysia. However, they performed no significant
biochemical tests to distinguish E. papayae and E. mallotivora, which are closely related species. They
relied on two basic biochemical tests (oxidase and catalase) and the sequence similarity of the 16S
rRNA gene to confirm E. papayae as a causal agent of papaya dieback in Malaysia. E. papayae was
first reported by Gardan et al. [5] as the causal organism of papaya bacterial canker in the Caribbean
region. However, the advanced stage of papaya dieback reported by Maktar et al. [4] had no canker
symptom. Thereafter, no additional information on further occurrence of the disease has been reported
until our finding, reported here, of E. mallotivora as a causal organism for this disease.
The aim of this study was, firstly, to isolate and identify the papaya dieback pathogen, and
subsequently, to confirm its pathogenicity through Koch’s postulate. By combining phenotypic and
genotypic information, we confirmed E. mallotivora as the causal agent of papaya dieback disease in
Peninsular Malaysia.
2. Results and Discussion
Gram-negative and rod-shaped bacteria with a peritrichous flagella arrangement were consistently
isolated from water-soaked lesions (Figure 1). The isolate from infected tissues produced hyaline
colonies on Luria Bertani (LB) agar, typically with slow growth (2–3 days incubation). Colonies were
creamy to white in color on King’s B [6] agar after 2–3 days incubation and on YBGA (0.7% yeast
extract, 0.7% bactopeptone, 0.7% glucose and 1.5% agar, pH 7.2) medium after 3–4 days incubation at
25 ° No water-soluble or non-water-soluble pigments were produced on King’s B medium.
C.
Biochemical analysis revealed that the strains are catalase positive and oxidase negative with the ability
to form reducing compounds from sucrose. In addition, the strains produced acid from D-mannitol and
utilized both lactate isomers, D- and L-lactate, which is consistent with the results reported by Goto [7]
and Gardan et al. [5] for E. mallotivora. In contrast, E. papayae was unable to produce acid from
mannitol, DL-lactate and sucrose-reducing compounds [5]. The apparent phenotypic characteristics of
E. papayae and E. mallotivora are summarized in Table 1.](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)